Spectroscopic signals from inherent molecular vibration offer a contrast mechanism for label-free imaging of biomolecules in cells and tissues. Accordingly, vibrational imaging of deep tissues holds great potential for in situ diagnosis based on the disrupted molecular mechanism in human diseases. However, vibrational imaging of deep tissues has been a formidable challenge due to tissue absorption and scattering of both incident photons and generated signals. For example, though coherent Raman scattering microscopy has allowed fast vibrational imaging, its penetration depth is limited to ˜100 μm because the signal is generated by ballistic photons under a tight focusing condition.
As a molecular and functional imaging modality, photoacoustic tomography (PAT) has demonstrated the imaging capability of several centimeters deep into biological tissues. In PAT, pulsed light is used to induce optical absorption inside a tissue by diffused photons. Part of the absorbed energy is converted into heat, which raises the temperature of the absorbed region on the order of mK. This sudden temperature change then creates pressure transients and subsequent generation of photoacoustic (PA) waves detectable by an ultrasonic transducer in real time. From the measured signal, the distribution of optical absorbers is reconstructed. The contrast mechanism in PAT is generally based on electronic absorption in the near infrared region extending up to 950 nm. For example, PAT imaging of hemoglobin and exogenous contrast agents such as dyes and nanoparticles has been reported Inherent molecular vibration offers a contrast mechanism for chemical imaging in a label free manner. In vibrational microscopy based on either infrared absorption or Raman scattering, the imaging depth is limited to the ballistic photon mean free path, which is a few hundred microns in a biological sample. Owing to much weaker acoustic scattering in tissues as compared to optical scattering, photoacoustic detection of harmonic molecular vibration has enabled significant improvement in imaging depth. In this method, optical absorption is induced by overtone transitions at near infrared wavelengths, such as the second overtone transition of C—H bond occurring around 1200 nm. Upon excitation, this vibrational energy quickly turns into heat, which leads to bond-selective photoacoustic signals. Overtone transitions have been used for intravascular photoacoustic imaging of lipid accumulation. The optical parametric oscillator currently used for PAT has been designed for excitation of hemoglobin and other contrast agents in wavelengths below 950 nm.
However, effective vibrational photoacoustic tomography (VPAT) has not yet been demonstrated, partly due to the unavailability of a laser source having sufficient energy for diffused photon excitation of harmonic vibration. There is, therefore, a need of an improved laser and improved VPAT system.